Cloning and Expression of cel B Cellobiohydrolase gene in Pichia pastoris

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Heydari Gharehsoo Fahimeh; MOSHTAGHI NASRIN; Meshkani Baratali; Malekzade Shafarudi Saeid

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Journal: GENETIC ENGINEERING AND BIOSAFETY JOURNAL Year:2019 | Volume:7 | Issue:2 Page(s): 139-151

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Title

Cloning and Expression of cel B Cellobiohydrolase gene in Pichia pastoris

Author(s)

Heydari Gharehsoo Fahimeh | MOSHTAGHI NASRIN | Meshkani Baratali | Malekzade Shafarudi Saeid | Issue Writer Certificate

Keywords

Cellobihydrolase gene

cel B

Consolidated Bioprocessing

yeast

Abstract

The cel B enzyme from Thermobifidia fusca, a CBHII, belongs to the family of cellulase B, which is highly resistant to heat. Due to the low level of production of this enzyme in this host, it cannot be used at industrial scale. For expression of cellobihydrolase in yeast, cel B gene in pSZ plasmid was amplified by PCR and then was cloned in to pPICZα A vector. Recombinant construct contains cel B gene transformed to E. coli Jm and recombinant were bacteria selected on LB medium with μ g/ml Belleomycin. Recombinant plasmid was linearized and then transformed in Gs and KM H yeast strains by electroporation method. Finally, the best concentration of methanol and the best day of expression of enzyme were determined by DNS method. The results in yeast indicated, the most CBH activity in GS and KM H strains and on PC substrate in C for h was and U ( mol/min)/ml, respectively. Only sample from high yield colony of GS strain for CBH expression was confirmed in SDS-PAGE and then the best methanol concentration and day for enzyme expression with same strain was obtained in th days after induction with three percent methanol. In this study, we did not optimize the codons of cel B gene for expression in P. pastoris. Thus, its decreased expression level and cellobiohydrolase activity may be due to codon usage in Pichia.

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APA: Copy

Heydari Gharehsoo, Fahimeh, MOSHTAGHI, NASRIN, Meshkani, Baratali, & Malekzade Shafarudi, Saeid. (2019). Cloning and Expression of cel B Cellobiohydrolase gene in Pichia pastoris. GENETIC ENGINEERING AND BIOSAFETY JOURNAL, 7(2 ), 139-151. SID. https://sid.ir/paper/270506/en

Vancouver: Copy

Heydari Gharehsoo Fahimeh, MOSHTAGHI NASRIN, Meshkani Baratali, Malekzade Shafarudi Saeid. Cloning and Expression of cel B Cellobiohydrolase gene in Pichia pastoris. GENETIC ENGINEERING AND BIOSAFETY JOURNAL[Internet]. 2019;7(2 ):139-151. Available from: https://sid.ir/paper/270506/en

IEEE: Copy

Fahimeh Heydari Gharehsoo, NASRIN MOSHTAGHI, Baratali Meshkani, and Saeid Malekzade Shafarudi, “Cloning and Expression of cel B Cellobiohydrolase gene in Pichia pastoris,” GENETIC ENGINEERING AND BIOSAFETY JOURNAL, vol. 7, no. 2 , pp. 139–151, 2019, [Online]. Available: https://sid.ir/paper/270506/en

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